Gillam E M, Guo Z, Martin M V, Jenkins C M, Guengerich F P
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia.
Arch Biochem Biophys. 1995 Jun 1;319(2):540-50. doi: 10.1006/abbi.1995.1329.
Cytochrome P450 (P450) 2D6 is the classic human liver debrisoquine 4-hydroxylase, the first human P450 for which genetic polymorphism was clearly demonstrated. We prepared 11 different constructs of P450 2D6, with modification at the N-terminus, for expression in Escherichia coli with the vector pCW. These varied considerably in levels of expression of apo- and holoprotein, with the best yield being obtained in a system in which much of the N-terminal hydrophobic segment was removed. Production of holoprotein was highly dependent upon the addition of delta-aminolevulinic acid and FeCl3 to cultures, even though heme production should not be limiting in this system. The expressed protein was not tightly bound to the "heavier" membrane fraction but did not appear to behave as a soluble protein either. A purification strategy was developed involving fractional centrifugation, Triton X-114 phase separation, and flavodoxin affinity chromatography, which led to recovery of apparently electrophoretically homogeneous protein in good yield. Purified P450 2D6 had the expected N-terminal amino acid sequence and catalytic activities toward debrisoquine (4-hydroxylation) and bufuralol (1'-hydroxylation). The availability of a ready source of the recombinant protein should facilitate physical as well as functional studies and antibody production for other uses.
细胞色素P450（P450）2D6是经典的人肝脏异喹胍4-羟化酶，是第一个其基因多态性得到明确证实的人P450。我们制备了11种不同的P450 2D6构建体，在N端进行了修饰，用于在大肠杆菌中与载体pCW一起表达。这些构建体在脱辅基蛋白和全蛋白的表达水平上有很大差异，在去除大部分N端疏水片段的系统中获得了最佳产量。全蛋白的产生高度依赖于向培养物中添加δ-氨基乙酰丙酸和FeCl3，尽管在该系统中血红素的产生不应受到限制。表达的蛋白没有紧密结合到“较重”的膜部分，但似乎也不是可溶性蛋白。开发了一种纯化策略，包括分级离心、Triton X-114相分离和黄素氧还蛋白亲和色谱，从而以良好的产量回收了明显电泳纯的蛋白。纯化的P450 2D6具有预期的N端氨基酸序列以及对异喹胍（4-羟化）和布非洛尔（1'-羟化）的催化活性。重组蛋白的现成来源的可用性应有助于进行物理和功能研究以及用于其他用途的抗体生产。
